Free thyroxine measurement in clinical practice: how to optimize indications, analytical procedures, and interpretation criteria while waiting for global standardization

Figures & data

Figure 1. Chemical structure of thyroid hormones.

Figure 2. Biosynthesis of thyroid hormones. (T4, thyroxine, T3, triiodothyronine, MCT8, monocarboxylate transporter 8; Tg, thyroglobulin; TPO, thyroid peroxidase; DIT, diiodotyrosine; MIT, monoiodotyrosine).

Figure 3. The log-linear inverse relationship between TSH and FT4. (TSH, thyroid stimulating hormone; FT4, free thyroxine). The blue line represents an approximate relationship between TSH and FT4. The dotted grey lines represent the normal values for TSH (horizontal) and FT4 (vertical).

Table 1. Clinical indications for FT4 measurement.

Indications
TSH suppressed	Differentiate subclinical from overt hyperthyroidism Assess the degree of hyperthyroidism
TSH increased	Differentiate subclinical from overt hypothyroidism Assess the degree of hypothyroidism
Hyperthyroidism treated with anti-thyroid drugs or radioiodine	Monitor the response (TSH unreliable in the initial months after therapy)
Pituitary diseases	Diagnosis of central thyroid dysfunctions (TSH unreliable)
Central hypothyroidism	Monitoring thyroxine therapy (TSH unreliable)

FT4: free thyroxine, TSH: thyroid-stimulating hormone.

Figure 4. Schematic of the four main immunoassay formats used to measure FT4. (A) In the two-step labeled T4 analog method, FT4 in the specimen interacts with anti-hormone antibodies on a solid support. Antibody binding sites are occupied by endogenous FT4. The specimen is then removed and any empty antibody binding sites are allowed to interact with labeled FT4 (“back-titration”). (B) In the one-step assay, FT4 in the specimen competes with the labeled analog for binding to the antibodies fixed on a solid phase. The amount of labeled hormone left after the final washing of the solid support is inversely proportional to the original concentration of free hormone in the sample. (C) In the one-step analog two-step incubation format, anti-T4 antibodies are first incubated with FT4 in the specimen. In the second incubation step, the labeled T4 analog and streptavidin-coated microparticles bind to the unoccupied anti-T4 antibody binding sites, forming an antibody-hapten complex, bound to the solid phase via interaction between biotin and streptavidin. The unbound labeled T4 analog is washed away and the signal recorded. (D) In the labeled antibody method, labeled T4 antibodies bind to either FT4 in the specimen or solid phase-bound T4. After a short incubation period, a wash is carried out and the signal is measured (FT4, free thyroxine; T4, thyroxine).

Table 2. Main analytical characteristics of the most used FT4 immunoassays as quoted by the manufacturers.

Manufacturer/assay	Principle of immunoassay	LOD (pmol/L)	LOQ (pmol/L)	Assay range (pmol/L)	Imprecision (CV %) (intra-assay; inter-assay; total)	RIs^† (pmol/L)
Abbott ARCHITECT Free T4	CMIA, two-step	3.60	5.15	5.15–64.35	2.3–5.3; ND; 3.6–7.8	9.01–19.05
Abbott Alinity i Free T4	CMIA, two-step	3.60	5.41	5.41–64.35	1.7–3.0; ND; 2.0–3.1	9.01–19.05
Beckman Coulter Access Free T4	CLIA, two-step	3.22	ND	3.22–77.20	1.8–4.4; 3.3–8.1; 4.3–9.2	7.86–14.41
Roche cobas Elecsys^® FT4 IV	ECLIA, one-step with two sequential incubations	0.5	1.3	0.5–100.0	1.6–5.0; 1.9–6.3; ND	11.9–21.6
Siemens Healthineers Centaur FT4	CLIA, one-step with labeled analog	1.3	ND	1.3–155	2.2–3.3; 2.3–4.0; 3.4–4.6	11.5–22.7
Siemens Healthineers Atellica IM FT4	CLIA, one-step with labeled analog	1.3	ND	1.3–154.8	1.2–4.7; 2.2–6.8; ND	11.5–22.7

CMIA: chemiluminescent microparticle immunoassay; CLIA: chemiluminescent assay; CV: coefficient of variation; ECLIA: electrochemiluminescence assay; FT4: free thyroxine; LOD: limit of detection; LOQ: limit of quantification; ND: not disclosed; RI: reference interval.

^†Reference intervals were calculated in a population of apparently healthy adult males and females. Information correct to May 2022.

Figure 5. Schematic depiction of the workflow for current (high-pressure) liquid chromatography-tandem mass spectrometry (LC-MS/MS) of FT4.

Table 3. Comparison of RIs of FT4 in adult populations on Abbott ARCHITECT platform.

Reference	Country	Number of patients and enrollment criteria	Partitioning criteria	Method for RI calculation	FT4 eRI (pmol/L)	FT4 mRI (pmol/L)
Quinn et al. [118]	China	414 (217 M; 197 F) Age range: 20–63 years Inclusion: TPOAb and TgAb negativity	Sex	Direct	Overall: 10.7–17.1	9.0–19.1 (De Grande et al. [121])
Schalin-Jäntti et al. [119]	Finland	511 (269 M; 242 F) Age range: 18–91 years Inclusion: TPOAb negativity	Age	Direct	Overall: 10.9–16.8
Milinkovic et al. [120]	Serbia	22,860 (11,440 M; 11,420 F) Age ≥18 years Inclusion: TPOAb and TgAb negativity	Age; sex	Indirect	Overall: 10.5–18.9 M: 10.8–18.3* F: 11.5–15.4*
Ehrenkranz et al. [122]	USA	69,223 (22,765 M; 46,458 F) Age range: 1–104 years Inclusion: 0.4 < TSH < 10 mIU/L Exclusion: personal or family history of thyroid disease; medications altering thyroid function; obstetric events within 9 months	Age; sex; daily and annual variations	Indirect	Overall: 9.8–19.1^†
Hickman et al. [123]	Australia	1177 (630 M; 547 F) Age range not specified Inclusion: TPOAb and TgAb negativity Exclusion: medications altering thyroid function	Age	Direct	Overall: 10.8–16.8
Barth et al. [117]	UK	214 (83 M; 131 F) Age range: 18–65 years Inclusion: TPOAb and TgAb negativity Exclusion: pregnant or lactating women; blood donors; individuals on medication or with long-term conditions such as diabetes	Sex	Direct	Overall: 10.6–15.5
Barhanovic  et al. [124]	Serbia	790 F Age range: 20–69 years Inclusion: 0.1 < TSH < 10 mIU/L Exclusion: personal or family history of thyroid disease; medications altering thyroid function; obstetric events within 9 months; breastfeeding	Age	Direct	20–29 y: 10.7–17.5
					30–39 y: 10.6–16.1
					40–49 y: 10.4–18.1
					50–59 y: 10.7–18.1**
					60–69 y: 11.0–19.1**

eRI: experimental reference interval, F: female, FT4: free thyroxine, M: male, mRI: manufacturer reference interval, RI: reference interval, TgAb: anti-thyroglobulin antibodies, TPOAb: anti-thyroid peroxidase antibodies, TSH: thyroid-stimulating hormone, UK: United Kingdom, USA: United States of America, y: years.

^†Mean of age and sex results.

*Statistically significant difference between M and F and between 31–40 and 41–50 years old.

**Statistically significant difference (higher values) in the two oldest cohorts compared with the younger groups.

Table 4. Comparison of RIs of FT4 in adult populations on Beckman Coulter platforms.

Reference	Country	Platform	Number of patients and enrollment criteria	Partitioning criteria	Method for RI calculation	FT4 eRI (pmol/L)	FT4 mRI (pmol/L)
Plouvier et al. [125]	France	Access 2	262 (72 M; 190 F) Age range: 18–65 years Selection according to NACB guidelines (Baloch et al. [126])	Sex	Direct	Overall: 8.5–14.9 M: 8.6–14.9* F: 8.4–14.6*	7.9–14.4 (De Grande et al. [121])
Wang et al. [127]	China	DxI 800	4095 (2439 M; 1656 F) Median age: 43 years Exclusion: overt diseases; pregnancy	Age; sex	Indirect	M: 8.9–14.7** F: 8.6–14.3**
Barth et al. [117]	UK	DxI 800	253 (105 M; 148 F) Age range: 18–65 years Inclusion: TPOAb and TgAb negativity Exclusion: pregnant or lactating women; blood donors; individuals on medication or with long-term conditions such as diabetes	Sex	Direct	Overall: 7.9–14.0
Li et al. [128]	China	DxI 800	313 (173 M; 140 F) Age range: 20–97 years Inclusion: TPOAb and TgAb negativity Exclusion: pregnant or lactating women; personal or family history of thyroid disease, abnormal liver function, tumor, or other endocrine and metabolic disease	Sex	Direct	Overall: 8.6–14.3 M: 9.0–14.5 F: 8.5–14.3
Wang et al. [129]	China	DxI 800	1828 Age range: 21–73 years Inclusion: TPOAb and TgAb negativity; sonographically assessed normal thyroid gland	Sex	Direct	Overall: 8.9–15 M: 9.1–15.2 F: 8.8–14.5

eRI: experimental reference interval, F: female, FT4: free thyroxine, M: male, mRI: manufacturer reference interval, NABC: National Academy of Clinical Biochemistry, RI: reference interval, TgAb: anti-thyroglobulin antibodies, TPOAb: anti-thyroid peroxidase antibodies, UK: United Kingdom.

*Statistically significant difference between M and F.

**Statistically significant difference between M and F; statistically significant difference between younger and older than 50 years old.

Table 5. Comparison of reference intervals of FT4 in adult populations on Roche Diagnostics platforms.

Reference	Country	Platform	Number of patients and enrollment criteria	Partitioning criteria	Method for RI calculation	FT4 eRI (pmol/L)	FT4 mRI (pmol/L)
Kratzsch et al. [130]	Germany	Elecsys	453 (279 M; 174 F) Age range: 18–68 years Inclusion: healthy blood donors, with normal thyroid gland assessed sonographically Exclusion: personal history of a thyroid disease	Age; sex	Direct	Overall: 12.8–20.4 M: 13.0–21.4* F: 12.2–20.0*	12.0–22.0 (De Grande et al. [121]) 12.0–22.0 (De Grande et al. [121])
Friis-Hansen et al. [131]	North Europe (DK, FI, NO, SE)	Modular E170	430 (226 M; 204 F) Age ≥18 years Inclusion: TPOAb and TgAb negativity Exclusion: chronic disease; blood donation; pregnancy or breastfeeding; intake of prescription drugs (except contraception pills)	Age; sex; country	Direct	Overall: 14.5–22.9
Takeda et al. [132]	Japan	Elecsys	878 (451 M; 427 F) Age range: 20–86 years Selection according to NACB guidelines (Baloch et al. [126])	Age; sex	Direct	Overall: 14.2–21.0
Chan et al. [133]	China	Modular E170	157 (66 M; 91 F) Age ≥18 years Inclusion: TPOAb and TgAb negativity Exclusion: consumption of tobacco and alcohol; acute, chronic, or congenital illnesses and/or infections; history of malignancies; use of herbs or vitamin supplements; limb amputation; overnight shift duties; heavy manual workers; pregnancy, recent miscarriage, or menstrual disturbance in female participants; personal or family history of thyroid disease; medications	Age; sex	Direct	Overall: 13.2–20.7 M: 13.5–21.3* F: 12.6–19.7*
Yoshihara  et al. [134]	Japan	cobas	1388 (198 M; 1190 F) Age range: ≥20 years Inclusion: normal thyroid gland assessed sonographically; negativity of anti-thyroid antibodies Exclusion: personal or familiar history of thyroid disorders; pregnant women; admission to hospital within the previous month; medication that might affect thyroid function; history of extrathyroidal malignancy; collagen disease; diabetes mellitus; neurologic disease; hepatic disorder; cardiovascular disease; chronic kidney disease	Age; sex	Direct	Overall: 11.7–20.0 M: 12.9–21.1 F: 11.6–19.7
Amouzegar  et al. [135]	Iran	cobas	2199 (953 M; 1246 F) Age ≥20 years Selection according to NACB guidelines (Baloch et al. [126])	Age; sex	Direct	Overall: 11.7–19.9 M: 12.2–20.6* F: 11.5–19.3*
Marwaha et al. [136]	India	Elecsys	1916 (916 M; 1000 F) Age ≥18 years Inclusion: normal thyroid gland assessed sonographically; TPOAb negativity Exclusion: history of thyroid disease; family history of thyroid disease; use of medications known to interfere with thyroid function; systemic illness; goiter	Age; sex	Direct	Overall: 10.1–24.8**
Fontes et al. [137]	Brazil	Modular E170	1200 (600 M; 600 F) Age ≥18 years Exclusion: past or present thyroid disease; thyroid surgery; family history of thyroid disease; TSH < 0.1 mIU/L or > 10.0 mIU/L; goiter; smoking; medicines known as possible analytical or physiological interference on measurement of TSH or FT4 in the past 3 months	Age; sex	Direct	M: 1.9–21.9 F: 1.9–24.5
Sriphrapradang et al. [138]	Thailand	cobas e 411	1947 (1047 M; 900 F) Age ≥14 years Exclusion: pregnant women; history of thyroid disorder; moderate to severe illness; medications affecting thyroxine-binding proteins or thyroid function	Age; sex; residential region	Direct	Overall: 12.4–22.9
Mirjanic-Azaric et al. [139]	Serbia	cobas	227 (73 M; 154 F) Age range not specified Selection according to NACB guidelines (Baloch et al. [126])	Age; sex	Direct	Overall: 12.3–20.0*
Park et al. [140]	Korea	cobas	5987 (3104 M; 2883 F) Age ≥10 years Inclusion: TPOAb negativity Exclusion: thyroid disease; family history of thyroid dysfunction; current pregnancy	Age; sex	Indirect	Overall: 11.8–20.6***
Barth et al. [117]	UK	cobas	233 (92 M; 141 F) Age range: 18–65 years Inclusion: TPOAb and TgAb negativity Exclusion: pregnant or lactating women; blood donors; individuals on medication or with long-term conditions such as diabetes	Sex	Direct	Overall: 12.5–19.6

DK: Denmark, eRI: experimental reference interval, F: female, FI: Finland, FT4: free thyroxine, M: male, mRI: manufacturer reference interval, NO: Norway, RI: reference interval, SE: Sweden, TgAb: anti-thyroglobulin antibodies, TPOAb: anti-thyroid peroxidase antibodies, TSH: thyroid-stimulating hormone.

*Statistically significant difference between M and F.

**Statistically significant difference between 18–30 years and >60 years in females and 18–30 years and >71 years in males.

***Statistically significantly decrease in FT4 with age.

Table 6. Comparison of reference intervals of FT4 in adult populations on the Siemens ADVIA Centaur platform.

Reference	Country	Number of patients and enrollment criteria	Partitioning criteria	Method for RI calculation	FT4 eRI (pmol/L)	FT4 mRI (pmol/L)
Reix et al. [141]	France	379 (192 M; 187 F) Selection criteria according to NACB guidelines (Baloch et al. [126])	Age; sex	Direct	Overall: 10.5–18.9	11.5–22.7 (De Grande et al. [121])
Wang et al. [142]	China	211 (94 M; 117 F) Selection criteria according to NACB guidelines (Baloch et al. [126])	Age; sex	Direct	Overall: 11.7–18.9*
Cai et al. [143]	China	717 (330 M; 387 F) Selection criteria according to NACB guidelines (Baloch et al. [126])	Age; sex	Direct	Overall: 11.0–20.4
					M: 11.4–21.1*
					F: 10.9–19.7*
Hoermann et al. [144]	Germany	271 (68 M; 203 F) Exclusion: hypothalamic/pituitary diseases; pregnancy; use of anti-thyroid drugs or thyroid replacement regimes	Age; sex	Direct	Overall: 11.1–17.3
Barth et al. [145]	UK	721 (A: 219; B: 222; C: 280) A: Redundant serum samples from primary care B: Repetition of series A after an interval of 12 months C: Healthy individuals	Not applied	Indirect (A and B); direct (C)	Overall: 10.0–20.0
					A: 10–18.1
					B: 11.1–20.6
					C: 11.8–19.2
Barth et al. [117]	UK	253 (99 M; 154 F) Age range: 18–65 years Inclusion: TPOAb and TgAb negativity Exclusion: pregnant or lactating women; blood donors; individuals on medication or with long-term conditions such as diabetes	Sex	Direct	Overall: 11.8–19.0
Zou et al. [109]	China	20,303 (10,170 M; 10,133 F) Median age: 37 years Healthy individuals extracted from the hospital and laboratory information system	Age; sex	Indirect	Overall: 12.2–20.1
					M: 12.8–20.6*
					F: 11.9–18.9*

eRI: experimental reference interval, F: female, FT4: free thyroxine, M: male, mRI: manufacturer reference intervals, NACB: National Academy of Clinical Biochemistry, RI: reference interval, TgAb: anti-thyroglobulin antibodies, TPOAb: anti-thyroid peroxidase antibodies, UK: United Kingdom.

*Statistically significant difference between M and F.

Table 7. Comparison of RIs of FT4 in elderly populations using different analytical platforms.

References	Analytical platform	Age range (years)	Number of patients	FT4 cRI (pmol/L)	FT4 eRI (pmol/L)
Ehrenkranz et al. [122]	Abbott ARCHITECT	61–80 >80	61–80: 10,790 >80: 1931	ND	61–80: 9.7–19.3 >80: 9.7–19.6
Barhanovic et al. [124]	Abbott ARCHITECT	60–69	130 F Routine tests over two consecutive years as a part of their health checkup	9.5–19.0	11.01–19.08
Fontes et al. [137]	Roche cobas Elecsys	≥60	360 Routine tests for which evaluation of thyroid function and / or autoimmunity was not required	11.6–ND	9.0–21.9
Yeap et al. [152]	Roche cobas Elecsys	70–89	411 Apparently healthy men	10.0–23.0	12.1–20.5

cRI: conventional reference interval as established by local clinical laboratory protocols, eRI: experimental reference interval, F: female; FT4: free thyroxine, M: male, ND: not disclosed.

Figure 6. FT4 concentrations during pregnancy and post-partum. Error bars represent the 5^th and 95^th percentiles per trimester. Dashed lines represent non-pregnant lower and upper reference limits. (Green, 1^st tertile; violet, 2^nd tertile; grey, 3^rd tertile; *p < 0.0001 compared with Tr1; **p < 0.01 compared with Tr1; NS, not statistically significant compared with Tr1m; FT4, free thyroxine; NS, non-significant; Tr1, trimester 1; Tr2, trimester 2; Tr3, trimester 3; TSH, thyroid-stimulating hormone; PP, post-partum).

Table 8. Comparison of gestational RIs of FT4 using different platforms, in studies published from January 2012 to May 2022 including at least two different trimesters of pregnancy.

Manufacturer/ analytical platform	Reference	Country	FT4 RIs (2.5–97.5)
			1^st trimester (No. samples)	2^nd trimester (No. samples)	3^rd trimester (No. samples)	NP (No. samples)
Abbott ARCHITECT System	Quinn et al. [179]	Mexico	9.7–17.9 (165)	9.5–16.7 (181)	8.4–14.4 (211)	10.7–17.6 (104)
	Shen et al. [180]	China	10.9–17.7 (365)	9.3–15.2 (346)	7.9–14.1 (480)	9.2–16.9 (360)
	Fan et al. [175]	China	NS: 7.2–19.8 (140) S: 12.8–18.6 (200)	NS: 9.8–17.7 (184) S: 10.6–15.3 (200)	NS: 9.8–17.9 (123) S: 9.6–14.4 (200)	7.5–21.1^†
	Liu et al. [177]	China	12.4–19.1 (312)	9.9–18.1 (304)	9.1–14.9 (331)	12.3–18.9 (150)
	Akarsu et al. [174]	Turkey	10.3–18.1 (945)	10.3–18.2 (1120)	10.3–17.9 (395)	10.7–18.2 (220)
	Ho et al. [166]	Singapore	11.4–19.5^‡ (561)	10.1–15.4^§ (557)	9.5–14.9^‖ (560)	ND
	Ollero [178]	Spain	10.9–16.0 (288)	10.6–15.4 (252)	8.6–13.6 (236)	ND
	Yang et al. [181]	China	11.7–19.7 (41,634)	ND	9.1–14.4 (41,634)	ND
	Yuen et al. [182]	Hong Kong	10.5–19.4	10.5–19.4	9.5–15.3	1^st T: 10.9–17.7
	Hernandez et al. [176]	Spain	10.4–16.0 (270)	8.4–12.7 (212)	8.2–12.5 (211)	9.0–19.0*
Beckman Coulter DxI 800	Ekinci et al. [162]	Australia	5.9–15.6 (129)	4.9–11.3 (84)	4.4–11.2 (71)	3.8–6.0^† (70)
	Zhang et al. [193]	China	8.7–15.2 (1521)	7.1–13.6 (1102)	3.2–12.0 (120)	ND
	Liu et al. [177]	China	9.1–15.7 (312)	6.6–13.5 (304)	5.9–12.8 (331)	9.1–15.2 (150)
	Sun et al. [192]	China	9.0–15.1 (1954)	6.8–11.3 (2839)	6.7–11.4 (2056)	9.4–15.9 (646)
	Kim et al. [189]	Korea	10.8–18.4 (135)	8.8–15.6 (143)	8.6–14.5 (139)	ND
	Yuen et al. [182]	Hong Kong	8.4–16.2 (524)	7.8–14.4 (524)	6.7–11.3 (524)	1^st T: 6.7–14.1
Roche Diagnostics Modular E170 cobas e 601 cobas e 602 Elecsys 1010	Moon et al. [190]	South Korea	10.7–21.2 (120)	9.1–15.7 (211)	8.4–14.6 (134)	12.1–19.3 (206)
	Joosen et al. [172]	The Netherlands	11.7–20.0 (97)	9.3–14.2 (94)	8.1–14.9 (93)	10.8–18.2^¶ (93)
	Sekhri et al. [191]	India	9.8–18.5 (86)	8.5–19.4 (86)	7.4–18.3 (86)	10.9–22.1 (124)
	Liu et al. [177]	China	13.1–22.2 (312)	9.8–18.9 (304)	8.7–15.4 (331)	13.5–20.0 (150)
	Zhang et al. [194]	China	SLRI group: 13.4–19.0** (99) CSRI group: 13.1–20.3 (957)	SLRI group: 10.5–16.2 (99) CSRI group: 9.2–17.7 (252)	SLRI group: 7.5–15.0 (99) CSRI group: 6.9–13.4 (91)	19.5–19.6 (301)
	Fan et al. [175]	China	NS: 13.4–22.5 (140) S: 13.0–20.0 (200)	NS: 10.0–17.8 (184) S: 10.4–16.0 (200)	NS: 8.7–14.8 (123) S: 9.5–14.6 (200)	12.0–22.0^†
	Khalil et al. [188]	United Arab Emirates	11.7–20.4 (136)	9.3–17.2 (146)	8.7–15.3 (132)	ND
	Donovan et al. [184]	Canada	11.0–19.2 (124)	10.5–18.2 (140)	9.0–16.1 (142)	1^st T: 12.1–19.6^†
						2^nd T: 9.6–17.0^†
						3^rd T: 8.4–15.6^†
	Zhou et al. [195]	China	11.5–21.5 (1183)	9.9–18.7 (1729)	8.8–15.2 (148)	1^st T: 11.8–18.4^†
						2^nd T: 11.6–17.4^†
						3^rd T: 9.7–15.1^†
	Yuen et al. [182]	Hong Kong	11.4–24.5	10.1–22.2	9.0–17.0	1^st T: 2.1–19.6^†
	Hernandez et al. [176]	Spain	11.5–19.1 (270)	9.7–14.7 (212)	8.9–14.5 (211)	12.0–22.0^†
	Bunch et al. [183]	USA	12.0–18.5 (453)	10.2–16.6 (479)	ND	ND
Siemens Healthineers ADVIA Centaur	Duan et al. [185]	China	12.3–18.9 (963)	11–15.5 (981)	9.5–16.3 (792)	ND
	Han et al. [186]	China	11.8–18.4 (188)	11.6–17.4 (133)	9.7–15.1 (157)	11.5–22.7^†
	Zhang et al. [196]	China	13.9–26.5 (288)	12.3–19.3 (255)	11.4–19.2 (262)	13.0–22.2 (282)
	Yuen et al. [182]	Hong Kong	11.3–20.3	10.9–19.4	10.1–16.0	ND
	Huang et al. [187]	China	11.9–18.8 (8053)	11.9–18.2 (8036)	10.2–17.4 (7612)	16.1–20.0 (8646)

CSRI: cross-sectional reference interval, FT4: free thyroxine, No.: number, ND: not disclosed, NP: non-pregnant, NS: non-sequential, PP: post-partum, S: sequential, SLRI: self-sequential longitudinal reference interval, T: trimester of pregnancy, TSH: thyrotropin.

^†Manufacturer Reference Intervals.

^‡9–14 weeks.

^§18–22 weeks.

^‖28–32 weeks.

^¶PP.

*Compared with the CSRI group, p < 0.05.

**Compared with the CSRI group, p < 0.01.

Figure 7. Age-dependent scatter plot of FT4 concentration, stratified by sex. (FT4, free thyroxine).

Table 9. Comparison of RIs of FT4 for children aged 1–18 years using different platforms, in studies published from January 2012 to May 2022.

Manufacturer/ Analytical platform	Reference	Country	Age group	Sample size (No.)	FT4 RIs (2.5–97.5 percentile)
Abbott ARCHITECT System	Aldrimer et al. [215]	Sweden	0.5–12 y	471	10.8–16.4
			13–18 y	215	10.2–15.5
	Radicioni et al. [219]	Italy	6.2–12.1 y 9.6–17.9 y	72 368	13.1–20.6 10.9–19.1
	Bailey et al. [211]	Canada	1–<19 y	952	11.4–17.6
	Bokulic et al. [218]	Croatia	1–<19 y	241	10.5–15.9
	Argente del Castillo et al. [217]	Spain	1–9 y	531	11.1–15.4
			10–13 y	577	9.9–14.9
			14–15 y	473	9.9–14.2
Beckman Coulter DxI 800	Romero et al. [220]	Mexico	13–18 m	47	14.0–35.1
			19–23 m	48	16.0–33.0
			2–<3 y	73	15.6–30.4
			3–<6 y	62	14.0–28.2
	Karbasy et al. [212]	Canada	3–<19 y	455	7.9–13.6
	Adeli et al. [209]	Canada	1–<19 y	982	13.0–21.0
Roche Diagnostics Modular E170 cobas e 601 cobas e 602 Elecsys 1010	Iwaku et al. [222]	Japan	4–6 y	43	14.4–21.5
			7–8 y	39	13.8–20.7
			9–10 y	51	12.4–20.6
			11–12 y	61	13.1–19.6
			13–14 y	72	19.4–19.6
			15 y	50	12.2–19.7
	Bohn et al. [150]	Canada	1–<19 y	982	13.0–21.0
	Gunapalasingham et al. [221]	Denmark	6–18.9	2407 (976 M; 1435 F)	ND
			6–9.9	866 (395 M; 471 F)	10.9–18.4
			10.0–14.9	1021 (605 M; 416 F)	10.0–17.8
			15.0–18.9	524 (165 M; 359 F)	10.2–17.8
Siemens Healthineers Advia Centaur	Strich et al. [224]	Israel	1–5 y	2722	11.0–19.0
			6–10 y	3452	11.3–18.7
			11–14 y	3429	10.5–17.9
			15–18 y	1019	10.4–18.0
	Loh et al. [223]	Singapore	7–10 y	NS	10.9–20.6
			10.1–12 y		10.2–20.1
Siemens Healthineers Atellica	Bohn et al. [214]	Canada	1–<19 y	810	13.4–21.1

FT4: free thyroxine, m: months, No.: number, ND: not disclosed, NS: not specified, RI: reference interval, y: years.

Table 10. Comparison of RIs of FT4 for infants aged 0–12 months using different platforms, in studies published from January 2012 to May 2022.

Manufacturer/ analytical platform	Reference	Country	Age group	Sample size (No.)	FT4 Reference Intervals (2.5–97.5 percentile)
Abbott ARCHITECT System	Bailey et al. [211]	Canada	5–14 d	66	13.5–41.3
			15–29 d	55	8.7–32.5
			30 d–11.9 m	270	11.4–21.9
	Bokulic et al. [218]	Croatia	2–15 d	68	11.8–28.0
			15 d–<12 m	54	11.3–18.9
Beckman Coulter DxI 800	Romero et al. [220]	Mexico	1 d–<1 m	47 (25 M; 22 F)	8.6–34.6 (8.4–30.6 M; 11.3–35.4 F)
			1 m–<6 m	76 (42 M; 34 F)	8.5–15.6 (7.8–15.4 M; 8.6–17.2 F)
			6–<12 m	52 (30 M; 22 F)	8.2–14.4 (8.1–12.1 M; 8.5–14.7 F)
	Karbasy et al. [212]	Canada	0–<20 d	80 (40 M; 40 F)	17.4–57.7
			20 d–3 y		9.5–17.8
	Adeli et al. [209]	Canada	0–<20 d	40	17.4–57.7^†
			20 d–3 y	215	9.52–17.8^†
	Jayasuriya et al. [229]	Australia	0–24 h	25	15.3–43.6
			25–48 h	51	14.7–53.2
			49–72 h	71	16.5–45.5
			73–96 h	86	17.8–39.4
			97–120 h	63	15.3–32.1
			121–144 h	32	14.5–32.3
			145–168 h	62	13.9–30.9
	Aktas et al. [234]	Turkey	4–7 d	482	18.66 ± 4.24^‡
			8–14 d	131	16.73 ± 2.57
			15–22 d	57	14.93 ± 2.83
			23–30 d	87	14.28 ± 3.86
	Wong et al. [237]	Malaysia	14–21 d	513	11.1–21.0
			22–30 d	66	10.1–19.6
Roche Diagnostics Modular E170 cobas e 601 cobas e 602 Elecsys 1010	Mutlu et al. [238]	Turkey	1 d	29	15.4–33.6
			3 d	39	15.4–42.5
			5 d	28	13.6–29.6
			7 d	30	14.5–34.6
			10 d	82	15.2–32.1
			14 d	22	14.5–28.7
			28 d	19	15.8–24.9
	Omuse et al. [236]	Kenya	1–7 d	552	13.6–34.8
			8–15 d	145	13.5–30.2
			15–20 d	465	14.2–24.8
			23–30 d	167	13.3–23.4
Siemens Healthineers Advia Centaur	Loh et al. [223]	Singapore	0–1.0 w	NS	19.9–46.6
			1.1–2.0 w		17.2–33.1
			2.1–3.0 w		15.0–25.9
			3.1–4.0 w		13.22–21.8
			1.1–2.0 m		11.3–21.3
	Strich et al. [224]	Israel	1 d–<1 m	47	12.4–27.4
			1 m–<2 m	58	12.4–21.8
			2 m–<12 m	317	10.8–19.5

^†Mean ± SD.

^‡2SD from mean.

d: days; F: females; FT4: free thyroxine; h: hours; M: males; m: months; No.: number; ND: not disclosed; NS: not specified; RI: reference interval; SD: standard deviation; w: weeks.

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